Process of preparing a protease inhibitor

ABSTRACT

Process for preparing a protease inhibitor from animal pancreas, for instance hog and cattle pancreas, following extraction of insulin therefrom with a dilute mineral acid, such as phosphoric acid, comprising the steps of: 1. EXTRACTING MOIST CUT-UP PANCREAS MATERIAL WITH 60 TO 70 PERCENT AQUEOUS ETHANOL HAVING A PH of 10-13, 2. REMOVING THE ETHANOL FROM THE EXTRACT BY EVAPORATION UNDER DECREASED PRESSURE AND AT A TEMPERATURE NOT EXCEEDING 40* C., 3. cooling the remaining concentrated extract whereby lipoids present therein are separated out and removing the separated lipoids, 4. INTRODUCING A WATER-SOLUBLE INORGANIC SALT AND PREFERABLY AMMONIUM OR MAGNESIUM SULFATE, WHEREBY A PRECIPITATE COMPRISING AN PROTEIN COMPLEX IS SEPARATED OUT AND RECOVERING THE PRECIPITATE, 5. FORMING A SUSPENSION OF THE PRECIPITATE FROM STEP 4 IN WATER HAVING A PH of 5.5 to 7.5 and heating the suspension to a temperature of 50* to 80* C, introducing trichloracetic or sulfosalicylic acid into the suspension in a concentration of 2 to 3 weight percent, cooling the resulting mixture to a temperature of 15* to 20* C., whereby the protein is separated out as a precipitate and separating off said albumin precipitate, 6. ADJUSTING THE PH of the filtrate to a pH of 7.0 to 7.5, saturating the filtrate with sodium chloride in an amount of 25 to 30 g/100 ml filtrate and acidifying the saturated filtrate with hydrochloric acid to provide a pH of 1.0 to 3.0 whereby the inhibitor is separated out as a precipitate, 7. SUBJECTING THE PRECIPITATE FROM STEP 6 TO SUCCESSIVE WASHINGS WITH A BUFFERED SODIUM CHLORIDE SOLUTION WHOSE PH is successively increased from 1.7 to 9.0 and separately recovering the fractions from each washing, combining the fractions recovered in the pH range of 3.0 to 6.0, subjecting the combined fractions to deionization and recovering the pure inhibitor.

United States Patent Mansfeld et al.

[ June 13, 1972 [54] PROCESS OF PREPARING A PROTEASE INHIBITOR [72]Inventors: Viktor Mansfeld; Zdenek Padr, both of Prague, CzechoslovakiaSpofa, United Pharmaceutical Works, Prague, Czechoslovakia [22] Filed:May 13,1970

[21] Appl.No.: 37,007

[73] Assignee:

Primary Examiner-Albert T. Meyers Assistant ExaminerFrederick E. WaddellAttorney-Michael S. Striker 7] ABSTRACT Process for preparing a proteaseinhibitor from animal pancreas, for instance hog and cattle pancreas,following extraction of insulin therefrom with a dilute mineral acid,such as phosphoric acid, comprising the steps of:

l. extracting moist cut-up pancreas material with 60 to 70 percentaqueous ethanol having a pH of 10-13,

2. removing the ethanol from the extract by evaporation under decreasedpressure and at a temperature not exceeding 40 C.,

3. cooling the remaining concentrated extract whereby lipoids presenttherein are separated out and removing the separated lipoids,

4. introducing a water-soluble inorganic salt and preferably ammonium ormagnesium sulfate, whereby a precipitate comprising an protein complexis separated out and recovering the precipitate,

5. forming a suspension of the precipitate from step 4 in water having apH of 5.5 to 7.5 and heating the suspension to a temperature of 50 to 80C, introducing trichloracetic or sulfosalicylic acid into the suspensionin a concentration of 2 to 3 weight percent, cooling the resultingmixture to a temperature of to C., whereby the protein is separated outas a precipitate and separating off said albumin precipitate,

6. adjusting the pH of the filtrate to a pH of 7.0 to 7.5, saturatingthe filtrate with sodium chloride in an amount of to g/ l 00 ml filtrateand acidifying the saturated filtrate with hydrochloric acid to providea pH of 1.0 to 3.0 whereby the inhibitor is separated out as aprecipitate,

7. subjecting the precipitate from step 6 to successive washings with abuffered sodium chloride solution whose pH is successively increasedfrom 1.7 to 9.0 and separately recovering the fractions from eachwashing, combining the fractions recovered in the pH range of 3.0 to6.0, subjecting the combined fractions to deionization and recoveringthe pure inhibi- I01.

3 Claims, No Drawings PROCESS OF PREPARING A PROTEASE INHIBITOR Thisinvention relates to a process for manufacturing a protease inhibitorfrom animal and in particular from cattle and/or hog pancreas followingpreliminary extraction of the insulin therefrom with a dilute mineralacid.

As is known, natural protease inhibitors are present in plant and animalorganisms. From the therapeutic standpoint, only the inhibitors ofanimal origin are important. The inhibitor has been found and recoveredwith equal success from difierent animal organs such as pancreas, lung,parotid, liver, spleen and the like and also from body fluids such asblood, milk and urine. There are many types of protease inhibitors andthey can be distinguished one from the other by their chemicalproperties, molecular size or stability and in particular by theirspecific activity against the proteolytic enzymes, i.e., trypsin,chymotrypsin, kallikerein, thrombin, plasmin, etc.

In the majority of cases, the preparation of known therapeuticallyuseful protease inhibitor substances uses liver, pancreas or parotid asstarting material. The crude material as a rule is extracted withmethanol and the crude fraction which contains the inhibitorprecipitated with acetone. The high molecular protein is removed bytreatment with sulfosalicylic acid and the use of ion exchangers and theactive compound then isolated through precipitation with acetone.

In accordance with the instant invention, an advantageous process formanufacturing a protease inhibitor from cattle and/or hog pancreas isprovided comprising acidifying the minced pancreatic material with amineral acid, in particular phosphoric acid to a pH of 2.0 to 3.5 andextracting the acidified material with 70 percent ethanol. Under theseconditions, the inhibitor is not simultaneously extracted with theinsulin but remains in the minced pancreas material. This lattermaterial when treated in the manner now set out permits recovery of theinhibitor in maximal yields and in high purity and activity.

In accordance with the invention, the starting material, i.e., moistenedcut-up pancreas, is extracted with 60 to 70 percent aqueous ethanolhaving a pH of 10.0-13.0, the ethanol then driven off from the extractby evaporation under lowered pressure and at a temperature of at thehighest 40 C., the lipoids present are then separated from theconcentrated extract by chilling, from the thusly purified aqueousextract an protein complex in the form of a precipitate is separated byintroducing into the extract a soluble inorganic salt, preferablyammonium or magnesium sulfate, thereafter the protein complexprecipitate is isolated and converted into an aqueous suspension havinga pH of 5.5-7.5, from which at a temperature of 50-80 C. by treatmentwith trichloroacetic acid or sulfosalicylic acid in a concentration of2-3 weight percent and through a subsequent cooling down to atemperature of l5-20 C., the ballast albumin in the form of aprecipitate is separated ofi (this fraction is not further worked up),adjusting the filtrate which contains the freed inhibitor to a pH of 7.0to 7.5, saturating the filtrate with sodium chloride in an amount of tog/ 100 ml and acidifying the saturated fltrate with hydrochloric acid toa pH of 1.0 to 3.0 whereby the total of the free inhibitor is separatedout in the form of a precipitate from which an enriched inhibitor isisolated by successively washing with a buffered sodium chloridesolution whose pH value is continuously increased upwardly from 1.7 to9.0, combining the fractions recovered in the pH range of 3.0 to 6.0 andafter deionization working up this combined fraction to the pureinhibitor in the known manner through freeze drying or precipitation.

For adjusting the pH value of the aqueous ethanol to 10.0 to 13.0 foruse in the extraction of minced pancreatic starting material, there isused an alkali metal hydroxide or ammonium hydroxide.

As buffered sodium chloride solution, there can be used a 22 to 25percent sodium chloride solution in 0.01 M phosphoric acid whose pHvalue can be continuously increased upwardly through addition of sodiumhydroxide.

The process for the manufacture of a protease inhibitor in accordancewith the invention is under the consideration of the properties of theraw material so regulated as to form a single intermediate product withthe goal of obtaining the highest yield and the most pure end product.It has been experimentally proven that it is most advantageous in thefirst step to hold the concentration of the extraction agent in thegiven range. At a lower concentration of the ethanol than 60 percent,too much of the ballast material passes over into the extract whichmakes the further working up difiicult. At higher concentrations than 70percent, the protease inhibitor is in general not extracted. Onconcentration of the extract, i.e., removal of the ethanol, there isrecovered an aqueous solution from which the lipoid substances are bestseparated by cooling. From the thusly purified aqueous solution, thereis isolated by salting out an protein complex in the form of aprecipitate which precipitate contains the inhibitor in a bound form.The freeing of the inhibitor takes place in the following step whereinthe protein complex at a pH of 6.0 to 7.0 by treatment withtrichloracetic or sulfosalicylic acid at moderately high temperatures issplit. This step is most important of the entire process, and only bymaintenance of optimal conditions throughout this step, are satisfactoryresults obtained. After this step, the protein formed in the splittingof the complex is separated off. The filtrate is then further worked up,and by salting out with sodium chloride and acidifying with hydrochloricacid, a precipitate produced which contains the total of the freeinhibitor. This precipitate or intermediate product can be worked up toform the desired end product by subjecting the same to fractionationeither by controlled contacting with hydrochloric acid in the presenceof sodium chloride having a pH of 1.7 to 9.0, or by introducing theprecipitate into a column filled with a suitable neutral material forinstance kieselguhr and eluting with a sodium chloride solution in adilute phosphoric acid whose pH is continuously increased from 3.0 to9.0. In both cases, the fractions obtained in the pH range of 3.0 to 6.0are collected and after deionization further worked up to the pureinhibitor material in the known manner for instance by freeze drying orprecipitation with acetone. The recovered substance is thereafter workedup to form the therapeutic agents. The agents can be formulated forinstance as sublingual tablets or injection solutions.

The pancreatic protease inhibitor obtained in accordance with theinvention evidences in pharmacological application a highly specificactivity. In testing a conventionally prepared series of inhibitors, thepreparation of the invention is established to be a relatively activefibrinolysis inhibitor and this is true both in vitro and in vivo. Invitro, this effect was further evaluated by means ofthromboelastographic determinations of rabbit plasma fibrinolysisfollowing pre-activation with streptokinase and also in an artificialfibrinogenthrombin-fibrinolysin system. In both test procedures, thepreparation of the invention demonstrated the highest degree of activityas compared to the conventionally prepared inhibitors and produced noblood coagulation inhibiting activity as for instance does the soybeanisolated inhibitor. In vivo, the estimation was carried out again underthe use of the referredto thromboelastic graph technique and withstreptokinase pretreated rabbits. In further experiments in vivo, thetime of the inhibitor activity was followed after administration ofdifferent intravenous or subcutaneous doses whereby equally highactivities were demonstrated in each case for the substances of theinvention.

The preparations of the invention demonstrate further valuablepharmacological properties. Their toxicity is low, therapeutic doses donot influence either the blood pressure or respiration of theexperimental animal, the high degree of purity of this material, i.e.,the same has the properties of a low molecular peptide which forclosesagainst a sensitization reaction.

The protease inhibitors produced by the process of the invention aresuitable for use as therapeutic agents in different conditions which arecharacterized by a disturbed equilibrium of the fibrinolytic system asfor instance found in gynecological practice, in differentpost-operative conditions, in pancreatic disease and the like. Theagents can be administered orally in the form of sublingual tablets orparenterally, for instance intravenously, which latter form is preferredin acute conditions in which a rapid increase of the protease inhibitorlevel is desired.

Further details of the process of the invention can be seen from thefollowing examples. The same describe only a single possibility ofcarrying out the process, and are accordingly not to be considered asexhaustive or limiting. In Example 1, the inhibitor is isolated throughfractional precipitation while in Example 2, the successive dissolutiontechnique is used. In both cases, an essentially pure product isobtained.

EXAMPLE 1 The squeezed-out hog or cattle minced moist pancreas materialremaining after extraction of insulin, was further comminuted and thenmixed with a two-fold volume (calculated on the amount by weight) of 66percent aqueous ethanol to which an 0.5 to 20 percent sodium hydroxidesolution had been added. The extraction was carried out under intensivestirring for 3 hours at room temperature. After this time had elapsed,the liquid fraction was separated off by centrifugation and the solidfraction treated a second time in the same manner as just described. Theethanolic extracts were combined, filtered and concentrated in a vacuumevaporator at 35 to 40 C. The concentrated extract was allowed to standfor 1 hour at room temperature whereby the lipoid material present wasseparated out. The lipoid material was separated off, washed withlukewarm water, and the wash water added to the concentrated extract.The extract was reacted slowly and under stirring with solid ammoniumsulfate in an amount of 550 g/l extract. The precipitated material whichformed was isolated after addition of kieselguhr either by suctionfiltration or centrifugation. Thereafter the precipitate was suspendedin a five-fold volume of water (calculated on the weight of theprecipitate), stirred and the pH adjusted to a value of 6.0 to 7.0 bymeans of a 20 percent sodium hydroxide solution. The suspension wasthereafter heated to 50 C. and under strong stirring, reacted withtrichloracetic acid in the form of a concentrationed solution to a finalconcentration of 2.5 percent. Under constant stirring, the suspensionwas heated to 70 C. and maintained constant at this temperature for 5minutes. The suspension was then cooled down to 15 to 20 C., allowed tostand for l to 2 hours and the precipitate which formed separated byfiltration. The clear filtrate was brought to a pH of 7.5 with a 20percent sodium hydroxide solution and solid sodium chloride in an amountof 28 g/ 100 ml then added. The viscous turbid liquid which was formedwas slowly reacted under stirring with percent hydrochloric acid to a pHof 2.0. A voluminous precipitate separated out, which was separated offby suction filtering or centrifugation and in the moist state or afterdewatering with an organic water miscible solvent such as acetone orether worked up further.

The precipitate which contains the total free inhibitor was purified bydissolving in water having a pH of 7.5 to form a 5 percent solution, thesolution reacted with solid sodium chloride in an amount of 25 g/ 100 mlwhereby a weak turbidity set in. Under strong stirring, this mixture wasreacted slowly in dropwise fashion with 10 percent hydrochloric acid,whereupon a precipitate formed. After a pH value of 6.0 was reached, theprecipitate was centrifuged off (Fraction A), and further acid added tobeing the pH to 4.5. A further precipitate was formed which was alsoseparated by centrifugation (F raction B). Following furtheracidification to a pH of 3.0, a further precipitate was separated(Fraction C), and finally the pH brought to 1.5 whereby the totalremaining peptide fraction (Fraction D) was separated. The Fractions Aand D have only a weak inhibiting activity against trypsin. The mostactive fraction is Fraction B. An active portion of the inhibitor iscontained in Fraction C. The fractions A and D were combined and were ata later time, purified through further fractionation procedures.

The active fractions B and C were combined, desalted following solutionin water with the aid of ion exchangers and after the desalting of thesolution, the inhibitor was isolated by freeze drying or throughprecipitation with acetone and dehydration of the precipitate with watermiscible organic solvents. The activity of the recovered inhibitorpreparation amounted to 2,000 to 5,000 KlU/l mg/international kallikreinunits. The yield was 8 to 10 million KIU from l00 kg of the cut-uppancreatic starting material.

EXAMPLE 2 In the same manner as in Example 1, there were carried out theethanol extraction, separation of lipoids, and separation of highermolecular protein fractions with trichloracetic acid. The furtherworking up of the active precipitate, i.e., the fractionating of asodium chloride containing solution thereof with hydrochloric acid wascarried out through fractionating on a kieselguhr column whereby ahighly active, pure and colorfree substance was recovered.

Kieselguhr was stirred into a 0.01 M phosphoric acid solution whichcontained 22 to 25 percent sodium chloride and which had a pH of 1.7 andthe resulting suspension poured into a column having a 35 mm diameter.There was formed a 210 mm high kieselguhr column. There was thenintroduced into the column a suspension formed by mixing 2g of activeprecipitate with one-third the amount of original kieselguhr containingsuspension whereby the column was increased in height by about 70 mm.The column was washed through with 0.01 M phosphoric acid and theelution begun with the linear gradient i.e., with a 0.01 M phosphoricacid solution containing 22 to 25 percent sodium chloride (pH 1.7). ThepH of such solution was continuously increased upwardly to a value of9.0 by means of 10 percent soda iye. The flow velocity amounted to 2.5to 5.0 ml/min. The fractions were collected in 25 ml aliquots. Duringthe elution, the pH of the eluting agent was increased at constantconcentration of sodium chloride. The initial fractions up to pH 3.0were substantially inactive, the active peptide fraction flowed throughnext (controlled with Follin-reagent) up to a pH of 5.0-5.4. Thefollowing fractions were less active. The eluate containing the activefraction was worked up as in Example 1. The activity of the recoveredproduct amounted to 2,000 to 5,000 KIU/lmg.

Without further analysis, the foregoing will so fully reveal the gist ofthe present invention that others can by applying current knowledgereadily adapt it for various applications without omitting featuresthat, from the standpoint of prior art, fairly constitute essentialcharacteristics of the generic or specific aspects of this inventionand, therefore, such adaptations should and are intended to becomprehended within the meaning and range of equivalence of thefollowing claims.

What is claimed as new and desired to be protected by Letters Patent isset forth in the appended claims.

We claim:

1. Process for preparing in inhibitor for proteolytic enzymes selectedfrom the group consisting of trypsin, chymotrypsin, thrombin, plasminand kallikrein from the hog and cattle pancreas material remainingfollowing extraction thereof with a dilute mineral acid for recovery ofinsulin therefrom which comprises the steps of (a) extracting such moistcut-up pancreatic material with 60 to 70 percent aqueous ethanol havinga pH of l0l3, (b) removing the ethanol from the extract by evaporationunder decreased pressure and at a temperature not exceeding 40 C., (c)cooling the remaining concentrated extract whereby lipoids presenttherein are separated out and removing the separated lipoids, (d)introducing a water-soluble inorganic salt selected from the groupconsisting of ammonium sulfate and magnesium sulfate whereby aprecipitate comprising a protein complex is separated out and recoveringthe precipitate, (e) forming a suspension of the precipitate from step(d) in water having a pH of 5.5 to 7.5 and heating the suspension to atemperature of 50 to 80 C, introducing trichloracetic or sulfosalicylicacid into the suspension in a concentration of 2 to 3 weight percent,cooling the resulting mixture to a temperature of to C, whereby theprotein is separated out as a precipitate and separating off saidprotein precipitate, (f) adjusting the pH of the filtrate to a pH of 7.0to 7.5, saturating the filtrate with sodium chloride in an amount of tog/ 100 ml filtrate and acidifying the saturated filtrate withhydrochloric acid to provide a pH of 1.0 to 3.0 whereby the inhibitor isseparated out as a precipitate, and (g) subjecting the precipitate fromstep (f) to successive washings with a buffered sodium chloride solutionwhose pH is successively increased from 1.7 to 9.0 and separatelyrecovering the fractions from each washing, combining the fractionsrecovered in the pH range of 3.0 to 6.0, subjecting the combinedfractions to deionization and recovering the pure inhibitor from thedeionized solution by precipitation with acetone or by freeze drying.

2. The process of claim 1 wherein the pH of the aqueous ethanol used instep (a) is adjusted to 10.0 to 13.0 by addition of an alkaline agentselected from the group consisting of sodium-, potassiumand ammoniumhydroxide.

3. The process of claim 1 wherein said bufiered sodium chloride solutionused in step g) is a 22 to 25 percent sodium chloride solution in 0.01 Mphosphoric acid whose pH is increased by addition of sodium hydroxidethereto.

l i II t

1. EXTRACTING MOIST CUT-UP PANCREAS MATERIAL WITH 60 TO 70 PERCENTAQUEOUS ETHANOL HAVING A PH OF 10-13,
 2. REMOVING THE ETHANOL FROM THEEXTRACT BY EVAPORATION UNDER DECREASED PRESSURE AND AT A TEMPERATURE NOTEXCEEDING 40*C.,
 2. The process of claim 1 wherein the pH of the aqueousethanol used in step (a) is adjusted to 10.0 to 13.0 by addition of analkaline agent selected from the group consisting of sodium-, potassium-and ammonium hydroxide.
 3. The process of claim 1 wherein said bufferedsodium chloride solution used in step g) is a 22 to 25 percent sodiumchloride solution in 0.01 M phosphoric acid whose pH is increased byaddition of sodium hydroxide thereto.
 3. COOLING THE REMAININGCONCENTRATED EXTRACT WHEREBY LIPOIDS PRESENT THEREIN ARE SEPARATED OUTAND REMOVING THE SEPARATED LIPOIDS,
 4. INTRODUCING A WATER-SOLUBLEINORGANIC SALT AND PREFERABLY AMMONIUM OR MAGNESIUM SULFATE, WHEREBY APRECIPITATE COMPRISING AN PROTEIN COMPLEX IS SEPARATED OUT ANDRECOVERING THE PRECIPITATE,
 5. FORMING A SUSPENSION OF THE PRECIPITATEFROM STEP 4 IN WATER HAVING A PH OF 5.5 TO 7.5 AND HEATING THESUSPENSION TO A TEMPERATURE OF 50* TO 80*C, INTRODUCING TRICHLORACETICOR SULFOSALICYLIC ACID INTO THE SUSPENSION IN A CONCENTRATION OF 2 TO 3WEIGHT PERCENT, COOLING THE RESULTING MIXTURE TO A TEMPERATURE OF 15* TO20*C., WHEREBY THE PROTEIN IS SEPARATED OUT AS A PRECIPITATE ANDSEPARATING OFF SAID ALBUMIN PRECIPITATE,
 6. ADJUSTING THE PH OF THEFILTRATE TO A PH OF 7.0 TO 7.5, SATURATING THE FILTRATE WITH SODIUMCHLORIDE IN AN AMOUNT OF 25 TO 30 G/100 ML FILTRATE AND ACIDIFYING THESATURATED FILTRATE WITH HYDROCHLORIC ACID TO PROVIDE A PH OF 1.0 TO 3.0WHEREBY THE INHIBITOR IS SEPARATED OUT AS A PRECIPITATE,
 7. SUBJECTINGTHE PRECIPITATE FROM STEP 6 TO SUCCESSIVE WASHINGS WITH A BUFFEREDSODIUM CHLORIDE SOLUTION WHOSE PH IS SUCCESSIVELY INCREASED FROM 1.7 TO9.0 AND SEPARATELY RECOVERING THE FRACTIONS FROM EACH WASHING, COMBININGTHE FRACTIONS RECOVERED IN THE PH RANGE OF 3.0 TO 6.0, SUBJECTING THECOMBINED FRACTIONS TO DEIONIZATION AND RECOVERING THE PURE INHIBITOR.